The use of Bacillus anthracis in acts of terrorism and/or biological warfare is a demonstrated threat to U.S. security. The long-term objective of this proposal is to develop inhibitors against a new drug target, the anthracis enzyme nicotinamide adenine dinucleotide (NAD) synthetase, that are effective therapeutic agents for preventing and/or treating infections caused by B. anthracis. NAD synthetase catalyzes the last step in both the de novo and salvage pathways for the biosynthesis of NAD, an essential cofactor in energy metabolism. Since exogenous NAD cannot support bacterial growth due to insufficient cell membrane permeability/transport, inhibitors of NAD biosynthesis should be bacteriostatic and/or bacteriocidal. We have identified the first low micromolar inhibitors of Bacillus NAD synthetase, and these effectively inhibit the vegetative growth of Bacillus anthracis, and are bacteriocidal, at concentrations around 1 (mu/g/mL. We will now perform reiterative design, chemical synthesis, and in vitro analysis to develop mature lead compounds. The Specific Aims are: (1) Using parallel, solution phase synthetic chemistry, we will optimize inhibitory activities for existing classes of lead structures until low nanomolar inhibitors are obtained. (2) Determine the molecular mechanism by which existing lead synthetic compounds inhibit NAD synthetase. In addition to protein crystallography, we will: (a) Measure the Ki and evaluate the type of inhibition for synthetic inhibitors of NAD synthetase. (b) Characterize the enzyme homodimer/monomer equilibrium and modulation by inhibitors. (c) Perform photoaffinity labeling experiments using inhibitors containing a photoreactive alkylating group (azide). (3) Alternative structural templates will be developed as new NAD synthetase inhibitors. All synthetic compounds will be evaluated in high throughput screens as enzyme inhibitors (IC50 and/or Ki) and antibacterials (MIC). Selectivity for the inhibition of Bacillus NAD synthetase over the human (erythrocyte) enzyme will also be evaluated.